Name: quantifying single microvessel permeability in isolated blood-perfused rat lung preparation
Uploaded: 2014-06-30T14:00:00
Description: Watch this Scientific Journal Video about Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation at JoVE.com

该研究是由美国国立卫生研究院HL75503以KP支持。

对动物进行的所有实验均批准的田纳西大学健康科学中心大学的机构动物护理和使用委员会。 1，油管灌注大鼠肺制剂<ol><li>准备一个管道系统用聚乙烯管（＃18），用于血液灌注， 如图1连接的压力传感器（P23XL），并放置在显微镜载物台上的管件。 </li><li>设定水浴温度为37°C。 </li><li>连接肺入口，并与聚乙烯管暂时出口。 </li></ol><p class="jove_content" fo:keep-togethe...

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孤立的血液灌注肺标本加上实时成像提供了确定在单肺微血管通透性改变一个简单的工具。我们采用该方法来定义响应于LPS输注渗透性的变化。我们的数据清楚地表明，脂多糖注射引起的增加微血管通透性。此外，数据还表明，LPS诱导的通透性的改变是相似的在两个小静脉和毛细血管。因此，这种技术的主要优点是定义单个微血管的泄漏，以及一个全局血管网络的能力。鉴于这种技术也产生全球?...

在肺微血管通透性增加导致肺泡水肿和损害气体交换的发展，是急性肺损伤（ALI）1-3的一大特点。因此，血管通透性的估计是在确定肺损伤，并提出治疗措施疗效的程度很重要。重量分析诸如血液中的游离肺湿重与干重比和微血管过滤系数被广泛使用的方法来估算渗透性4,5。其它方法包括定量肺组织6-8中的放射性或荧光探针的保留。然而，上述方法需要肺组织样品的postexperiment加工朝向阐明的渗透率数据。此外，由于一动物只能用于单一治疗方案，大型动物数​​量，可能需要一个完整的研究。上述方法的一个共同特点是，它们确定平均血管通透性为所有的血管组织样本内。但是，它是公认的肺部微观和宏观血管表型不同9。因此，渗透性反应可能是异构的各种血管段以及9,10之间。因此，定量组织样本中的所有肺血管通透性均可能没有充分反映这种异质性。 在离体血液灌注肺标本，血管对肺表面可以通过一个堂堂正正的显微镜4,11,12可视化。这使得在单个容器的反应特征，因此，解决在应对13任异质性。此外，通过利用微血管的荧光成像，基于荧光测定法可并入。进一步，左心房微导管可被用于运载剂和荧光探针进入血管11,14。微导管限制了传递到小肺部区域，从而前冒充只有区域内的血管的灌注剂和荧光基团。这允许将用于单独的实验相同的肺中的多个小区域，导致所需的研究动物的整体减少。 即时成像能够在血管和微血管单隔离肺准备的荧光血管外动态变化的捕获。因此，对于像场中的每个微血管，输液荧光和冲刷的过程中发生变化的荧光可以被记录，并量化离线14。使用的最大和残余血管荧光值，摄像视场中的每个微血管通透性指数可以被确定。以确定响应于炎性或损害性药物的渗透性的变化，所需的剂可以首先给药，然后通透性指数来确定。此外，该图像字段可以设置为通过注入肺区域内微导管，从而使之在选择所需的血管网的高度灵活性。因此，孤立的血液灌注肺的准备以配合实时成像提供了一个有吸引力的实验模型来量化通透性单肺微血管。

<table border="0" cellpadding="0" cellspacing="0" width="740">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tygon Tubing</td>
			<td style="width:108px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:297px;">#18</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pressure Transducer</td>
			<td style="width:108px;">Data Sciences International</td>
			<td style="width:119px;">P23XL</td>
			<td>Need quantity 3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Butterfly Needle</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td align="right" style="width:119px;">450081</td>
			<td>21G</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peristaltic pump</td>
			<td style="width:108px;">Cole Parmer</td>
			<td style="width:119px;">Masterflex L/S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-90 tubing</td>
			<td style="width:108px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">427421</td>
			<td>30 cm needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-10 tubing</td>
			<td style="width:108px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">427401</td>
			<td>40 cm needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringe Pump</td>
			<td style="width:108px;">Braintree Scientific</td>
			<td style="width:119px;">BS8000</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">O-ring</td>
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			<td style="width:119px;"></td>
			<td>Custom made with a 20 mm diamter hole&#160; and a handle&#160; to secure o-ring to holder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Upright fluorescence microscope</td>
			<td style="width:108px;">Olympus America</td>
			<td style="width:119px;">BX61WI</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Image Acquisition Software</td>
			<td style="width:108px;">Molecular Devices</td>
			<td style="width:119px;">Metamorph</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FITC Dextran 20KD</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td>0.5 mg/ml (A dextran of different molecular size can be selected, if trial experiments indicate its suitability based on the calculated permeability index values)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lipopolysaccharide</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td>Serotype 0111:B4</td>
		</tr>
	</tbody>
</table>

quantifying single microvessel permeability in isolated blood-perfused rat lung preparation

分离的血液灌注的肺制剂被广泛用于可视化和单微血管定义信令。通过实时成像耦合此制备中，以确定在单个肺微血管通透性的改变成为可能。在这里，我们描述的步骤来隔离大鼠肺组织，并与自体血灌注他们。然后，我们概述的步骤注入通过微导管荧光团或代理人到一个小细胞肺癌区域。使用上述这些程序，我们确定通透性增加大鼠肺微血管针对细菌脂多糖注射。数据显示，脂多糖通过这两个小静脉和毛细血管微血管段增加液体泄漏。因此，该方法使得有可能以比较的血管段中的渗透性的反应，因此，限定在响应中的任何不均匀性。而常用的方法来定义肺通透性要求肺组织样品，进行后处理的使用实时成像消除这一要求作为显而易见的，从目前的方法。因此，隔离肺制剂结合实时成像提供了一些优于传统方法来确定肺微血管的通透性，又是一个简单的方法来制定和实施。

分离的血液灌注肺制剂连接到灌注管道和相关设备示于图2。出于演示的目的，我们使用了一个只SD大鼠，虽然本文所描述的程序，可以用任何鼠种使用。通过左心房微导管输注到达肺的一小区域。该注入区域可通过输注引起的变色（ 图3）来识别。作为定位于实时成像的肺制剂示于图4。 <img alt="图2" fo:content-wid...

Watch this Scientific Journal Video about Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation at JoVE.com

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

在离体血液灌注大鼠肺备量化单微血管通透性

孤立的血液灌注肺的准备，使可视化微血管网络上的肺表面是可行的。在这里，我们描述的方法用实时荧光成像量化单一微​​血管隔离肺的通透性。

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Research

JoVE Journal

Biology

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

标准化的制备小鼠肺组织单细胞悬浮液使用gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

科研

生物学

Preparation of Highly Coupled Rat Heart Mitochondria

高度耦合的大鼠心肌线粒体的制备

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

表征隔离，通风，和仪表小鼠肺灌注与脉动流

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

“体外骨骼肌微血管制备离体血管反应性的调查

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

测量渗透水渗透系数（P<sub&gt; F</sub&gt;）球形细胞：隔离植物原生质体为例。

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

单通道分析和钙成像在新鲜分离的肾小球足细胞

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

微灌技术在调查微血管渗透性的大鼠肠系膜条例

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

CD146的分离<sup&gt; +</sup&gt;从大鼠肺组织驻地肺间质基质细胞

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

The Mouse Isolated Perfused Kidney Technique

鼠标离体肾技术

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...